There is a critical need for better clinical treatments of craniofacial bone tissue defects. Lactoferrin (LF), a multifunctional moonlighting glycoprotein, is also a potent anabolic effector of the skeleton. LF's anabolic effect in bone morphongenesis is attributed to its strong dose-dependent proliferative and anti-apoptotic actions in osteoblastogenesis and inhibition of osteoclastogenesis. We hypothesize that an injectable gel can be produced from recombinant human LF (rhLF) and this biomaterial will possess osteogenic activities. We propose to first investigate the physical properties and characterize the rhLF gels to be further employed as a regenerative skeletal biomaterial. We will characterize the physical properties of the injectable rhLF gels based on swelling, mechanical strength, morphology and in vivo degradation to be further applied as an injectable biomaterial. We hypothesize that varying concentrations of the three gel components, horseradish peroxidase (HRP), modified rhLF and H2O2, will directly affect the water uptake, mechanical strength, morphology and in vivo degradation rates of the rhLF gels. rhLF gels will be subjected to rheological testing to assess the relative mechanical strengths at varying concentrations of the three gel components. The morphology of the gels will be visualized by scanning electron microscope. The in vivo degradation of the rhLF gels will be evaluated using a subcutaneous mouse model. Thus, the proposed studies are aimed to increase our understanding of the rhLF gel physical properties. Next, we plan to explore the biological effects of rhLF gel as a novel osteogenic biomaterial. The second specific aim will be to investigate the ability of rhLF gels to promote cell survival, proliferation and differentiation of murine preosteoblast cells. We propose to use the MC3T3 preosteoblastic cell line to evaluate the effects of rhLF gel in promoting cell survival, proliferation and differentiation in vitro. Cells will be added to the HRP and modified rhLF solution and the subsequent addition of H2O2 will produce MC3T3 cell - laden rhLF gels. Gelatin will be modified similar to rhLF to form cross-linked gels, which will serve as the control biomaterial in these studies. Cell survival will be assessed by quantification of live to dead cell ratio. Proliferation of the cells encapsulated in the rhLF gels will be determined through thymidine incorporation. Alizarin Red will be utilized to study the degree of mineralization after 21 days of culture. Additionally, osteocalcin, osteopontin and Runx2, alkaline phosphatase expression levels will be assessed to study the effects of the rhLF gel on cell differentiation. Th proposed study will increase our understanding of the biological effect of rhLF gel in modulating skeletal cells and further establish rhLF as an excellent candidate for a skeletal regenerative biomaterial. PUBLIC HEALTH RELEVANCE: Lactoferrin (LF), a protein native to the human body, has been demonstrated to possess multifold functions and is well recognized for its influential role in bone growth. The proposed study aims to develop a LF-based biomaterial gel and increase our understanding of the effect of LF gel on bone cells. The development of this novel, bioactive biomaterial for bone repair will address the need for better clinical treatments of craniofacial bone tissue defects.